Enhanced in vitro testing model for rapid risk assessment of ingested nanomaterials

C. Carnovale, D. Guarnieri, I. De Angelis, F. Barone, S. Sabella
Italian Institute of Technology,

Keywords: nanomaterial, risk assessment, toxicity, uptake, translocation


Following advances in the synthesis of manufactured nanomaterials, interest for their inclusion in various consumer products has increased. Due to various factors including their unique optical and antimicrobial properties, likely applications of nanomaterials include textiles, cosmetic formulations, novel materials for food storage and health supplements. Accompanying this progress is the increased likelihood of chronic exposure to nanomaterials, and inevitably their ingestion – whether intentional or not. The inherent properties of nanomaterials, including composition and structure are obvious factors which require consideration for their risk potential, however developments in the safety assessment of nanomaterials have highlighted the contribution of extrinsic environmental factors on the stability and behavior of MNMs in vitro/ vivo (e.g., variations in pH, salt and protein concentration). While there are a number of validated cellular assays and acellular in vitro digestion models in use, many lack consideration for the impact of extrinsic factors on nanomaterials. For the risk analysis of ingested materials, solubility and stability within biological fluids (such as saliva, gastric and duodenal juices) are key factors to be considered, however despite this knowledge, many current methods utilize pristine nanoparticles as test material for cells which are located at distal positions of the digestive system. Furthermore, many studies conducted previous explore only a single exposure scenario and are unable to adapt to realistic, chronic exposure conditions. Given the awareness that nanomaterials may undergo significant change during the digestion process, we have designed a novel testing approach to more realistically monitor the fate of nanomaterials within the human digestive system. The system couples two validated testing methods, taking advantage of their demonstrated applicability for the safety screening of nanomaterials, and their proven ability to generate accurate datasets. The technique combines a dynamic in vitro gastrointestinal simulation with a Caco-2 cell monolayer to mimic real life conditions, which occur at the nano-cellular interface following repeated ingestion of nanomaterials over a prolonged period. By subjecting the nanomaterials to a simulated digestion process, a more realistic sample is obtained to perform subsequent tests related to the uptake and toxicity of nanomaterials and translocation through the intestinal epithelial barrier. The testing model has been used to assess three reference nanomaterials with different dissolution properties chosen from the OECD Testing Programme and NANoREG project (FP7, GA 310584). Additionally, we show the wide range of applicability of the test, also used here to assess the behavior of new pharmaceuticals including nanoformulations, which are designed for oral delivery.